Recombinant Anti-CDC42 antibody [EPR15620] (ab187643)

Lanes 1-4: Merged signal (red and green). Green - ab187643 observed at 20 kDa. Red - loading control ab8245 observed at 37 kDa.

 ab187643 Anti-CDC42 antibody [EPR15620] was shown to specifically react with CDC42 in wild-type HEK-293T cells. Loss of signal was observed when knockout cell line ab266522 (knockout cell lysate ab256868) was used. Wild-type and CDC42 knockout samples were subjected to SDS-PAGE. ab187643 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 5000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

Immunocytochemistry/Immunofluorescence analysis of U937 (Human histiocytic lymphoma cell line) labelling CDC42 with purified ab187643 at 1/500. Cells were fixed with 4% PFA and permeabilized with 0.1% triton X-100. ab150077 Goat anti rabbit IgG (Alexa Fluor^® 488) at 1/1000 was used as the secondary antibody. Nuclei were counterstained with DAPI. PBS was used instead of the primary antibody as the negative control.

Lanes 1 - 4: Merged signal (red and green). Green - ab187643 observed at 20 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab187643 was shown to specifically react with CDC42 in wild-type HAP1 cells. No band was observed when CDC42 knockout samples were examined. Wild-type and CDC42 knockout samples were subjected to SDS-PAGE. ab187643 and ab8245 (loading control to GAPDH) were diluted at 1/5000 and 1/10,000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10,000 dilution for 1 hour at room temperature before imaging.

Lane 1 (input): HT-29 (human colorectal adenocarcinoma epithelial cell) whole cell lysate,10μg
Lane 2(+): HT-29 whole cell lysate
Lane 3(-): Rabbit monoclonal IgG (ab172730) instead of  ab187643 in HT-29 whole cell lysate

Ab187643 immunoprecipitating CDC42 in HT-29 whole cell lysate. Capture antibody was used at a 1:60 dilution. For western blotting, ab187643 was used as the primary antibody at a 1:1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution.

Blocking and diluting buffer: 5% NFDM/TBST

Intracellular Flow Cytometry analysis of HeLa cells (human cervix adenocarcinoma epithelial).Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. Primary antibody was used at a 1/120 dilution (red). A Goat anti rabbit IgG (Alexa Fluor^®488, ab150077) (1/2000 dilution) was used as the secondary antibody. Rabbit monoclonal IgG (ab172730) was used as the isotype control (black). Cell without incubation with primary antibody and secondary antibody (Blue). 

ab187643 staining CDC42 in Human lung carcinoma tissue sections by Immunohistochemistry (IHC-P- paraformaldehyde-fixed, paraffin-embedded sections). Antigen retrieval was by heat mediation using ab93684 (Tris/EDTA buffer, pH 9.0).  Samples were incubated with primary antibody at a 1/500 dilution. A ready to use Goat Anti-Rabbit IgG H&L (HRP) was used as the secondary antibody. Hematoxylin was used as a counter stain. Cytoplasmic staining on human lung carcinoma.

ab187643 staining CDC42 in Mouse colon tissue sections by Immunohistochemistry (IHC-P- paraformaldehyde-fixed, paraffin-embedded sections).  Antigen retrieval was by heat mediation using ab93684 (Tris/EDTA buffer, pH 9.0).  Samples were incubated with primary antibody at a 1/500 dilution. A ready to use Goat Anti-Rabbit IgG H&L (HRP) was used as the secondary antibody. Hematoxylin was used as a counter stain. Cytoplasmic staining on mouse colon.

ab187643 staining CDC42 in Rat colon tissue sections by Immunohistochemistry (IHC-P- paraformaldehyde-fixed, paraffin-embedded sections).  Antigen retrieval was by heat mediation using ab93684 (Tris/EDTA buffer, pH 9.0).  Samples were incubated with primary antibody at a 1/500 dilution. A ready to use Goat Anti-Rabbit IgG H&L (HRP) was used as the secondary antibody. Hematoxylin was used as a counter stain. Cytoplasmic staining on rat colon.

Immunohistochemical analysis of paraffin-embedded Human breast carcinoma tissue labeling CDC42 with ab187643 at 1/250 dilution, followed by prediluted HRP Polymer for Rabbit IgG. Counter stained with Hematoxylin.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.